This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Individuals who have been infected with HIV and remain healthy for over 10 years are considered long-term survivors. These asymptomatic individuals have a strong cell-mediated immune response that suppresses HIV replication. This activity is lost as the infected person advances to disease. The antiviral response is mediated by a secreted CD8+ cell antiviral factor (CAF) which blocks transcription via the HIV promoter. CAF is produced at low levels by CD8+ cells from these healthy infected individuals. The protein appears to be unlike any of the known cytokines, chemokines and human growth factors. CAF identification will most likely be achieved through protein purification and mass spectrometry.CD8+ cells from healthy HIV-infected individuals are grown in culture medium lacking serum and where possible, albumin. Fluids are assayed for antiviral activity by standard procedures in the laboratory. Active fluids are then concentrated and fractionated by various biochemical techniques including ion exchange, chromatography, and by sizing columns Fractions with enriched CAF activity are analyzed by 2-D gel and evaluated by mass spectrometry. By column chromatography, we have been able to isolate two fractions from a sizing column (TSK) which have anti-HIV activity. Each of them has a limited number of proteins, which are being further evaluated as possible candidates for CAF activity. We are conducting isotope labeling experiments in attempts to quantitate differences in proteins expressed in CAF+ and CAF- fluids. By further mass spectrometry, we hope to be able to identify the protein (CAF) that mediates this anti-HIV activity.
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